This invention relates to vascular endothelial growth factor. More specifically, this invention relates to oligonucleotides specific for vascular endothelial growth factor nucleic acid and useful treatment of disorders that are associated with neovascularization and angiogenesis, such as psoriasis.
Neovascular diseases of the retina such as diabetic retinopathy, retinopathy of prematurity, and age-related macular degeneration are a major cause of blindness in the United States and the world, yet the biochemical events responsible for these processes have not been fully elucidated.
Diabetic retinopathy is the leading cause of blindness among working age adults (20-64) in the United States (Foster in Harrison""s Principles of Internal Medicine (Isselbacher et al., eds.) McGraw-Hill, Inc., New York (1994) pp. 1994-1995). During the course of diabetes mellitus, the retinal vessels undergo changes that result in not only leaky vessels but also vessel drop out resulting in retinal hypoxia. The effects of these complications are hemorrhaging, xe2x80x9ccotton woolxe2x80x9d spots, retinal infarcts, and neovascularization of the retina resulting in bleeding and retinal detachment. If left untreated, there is a 60% chance of visual loss. Classic treatment for proliferative diabetic retinopathy is panretinal laser photocoagulation (PRP). However, complications can occur from panretinal laser photocoagulation such as foveal burns, hemorrhaging, retinal detachment, and choroidal vessel growth. Furthermore, other untoward effects of this treatment are decreased peripheral vision, decreased night vision, and changes in color perception (Am. J. Ophthamol. (1976) 81:383-396; Ophthalmol. (1991) 98:741-840). Thus, there is a need for a more effective treatment for diabetic retinopathy.
Retinopathy of prematurity (ROP) is a common cause of blindness in children in the United States (Pierce et al. (1994) Int. Ophth. Clinics 34:121-148). Premature babies are exposed to hyperoxic conditions after birth even without supplemental oxygen because the partial pressure of oxygen in utero is much lower than what is achieved when breathing normal room air. This relative hyperoxia is necessary for their survival yet can result in ROP. The blood vessels of the retina cease to develop into the peripheral retina resulting in ischemia and localized hypoxic conditions as the metabolic demands of the developing retina increase. The resulting hypoxia stimulates the subsequent neovascularization of the retina. This neovascularization usually regresses but can lead to irreversible vision loss. There are at least 10,000 new-cases per year with a worldwide estimate of 10 million total cases. At present, there is no effective cure for ROP. Two therapeutic methods, cryotherapy and laser therapy, have been used but are not completely effective and themselves cause damage to the eye, resulting in a reduction of vision (Pierce et al. (1994) Int. Ophth. Clinics 34:121-148). Many other antiangiogenic compounds have been tested, but no inhibition in retinal neovascularization has been reported (Smith et al. (1994) Invest. Ophthamol. Vis. Sci. 35:1442; Foley et al. (1994) Invest. Ophthamol. Vis. Sci. 35:1442). Thus, there is a need for an effective treatment for ROP.
Age related macular degeneration is one of the leading causes of blindness in older adults in the United States, and may account for up to 30% of all bilateral blindness among Caucasian Americans (Anonymous (1994) Prevent Blindness America). This disease is characterized by loss of central vision, usually in both eyes, due to damage to retinal pigment epithelial cells which provide physiological support to the light sensitive photoreceptor cells of the retina. In most cases there is currently no effective treatment. In approximately 20% of exudative cases that are diagnosed early, laser treatment can prevent further loss of vision; however, this effect is temporary (Bressler et al., Principles and Practices of Ophthamology (eds. Albert and Jakobiac), W.B. Saunders Co., Philadelphia, Pa.) (1994) Vol. 2 pp. 834-852). Thus, there is a need for a more effective and permanent treatment for age related macular degeneration.
Ocular neovascularization is also the underlying pathology in sickle cell retinopathy, neovascular glaucoma, retinal vein occlusion, and other hypoxic diseases. These eye diseases as well as other pathological states associated with neovascularization (i.e., tumor growth, wound healing) appear to have hypoxia as a common factor (Knighton et al. (1983) Science 221:1283-1285; Folkman et al. (1987) Science 235:442-446; Klagsbrun et al. (1991) Ann. Rev. Physiol. 53:217-239; Miller et al. (1993) Principles and Practice of Ophthamology, W.B. Saunders, Philadelphia, pp. 760; and Aiello et al. (1994) New Eng. J. Med. 331:1480-1487). Moreover, retinal neovascularization has been hypothesized to be the result of a xe2x80x9cvasoformative factorxe2x80x9d which is released by the retina in response to hypoxia (Michaelson (1948) Trans. Ophthamol. Soc. U. K. 68:137-180; and Ashton et al. (1954) Br. J. Ophthamol. 38:397-432). Recent experimental data show a high correlation between vascular endothelial growth factor expression and retinal neovascularization (Aiello et al. (1994) New Eng. J. Med. 331:1480-1487). Furthermore, elevated levels of vascular endothelial growth factor have recently been found in vitreous from patients with diabetes (Aiello et al., ibid.). Thus, this cytokine/growth factor may play an important role in neovascularization-related disease.
Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) is an endothelial cell-specific mitogen which supports angiogenesis in wound healing and development. In addition, VEGF has recently been shown to be stimulated by hypoxia and required for tumor angiogenesis (Senger et al. (1986) Cancer 46:5629-5632; Kim et al. (1993) Nature 362:841-844; Schweiki et al. (1992) Nature 359:843-845; Plate et al. (1992) Nature 359:845-848). It is a 34-43 kD (with the predominant species at about 45 kD) dimeric, disulfide-linked glycoprotein synthesized and secreted by a variety of tumor and normal cells. In addition, cultured human retinal cells such as pigment epithelial cells and pericytes have been demonstrated to secrete VEGF and to increase VEGF gene expression in response to hypoxia (Adamis et al. (1993) Biochem. Biophys. Res. Commun. 193:631-638; Plouet et al. (1992) Invest. Ophthamol. Vis. Sci. 34:900; Adamis et al. (1993) Invest. Ophthamol. Vis. Sci. 34:1440; Aiello et al. (1994) Invest. Ophthamol. Vis. Sci. 35:1868; Simorre-Pinatel et al. (1994) Invest. Ophthamol. Vis. Sci. 35:3393-3400). In contrast, VEGF in normal tissues is relatively low.
VEGF has also been shown to play a major role in other diseases associated with the aberrant angiogenesis, including tumor development and skin disorders. Conditions associated with such irregularities include cancer, rheumatoid arthritis, the bullous diseases (including bullous pemphigoid, dermatitis herpetiformis, and erythema multiforme), and psoriasis.
Psoriasis is a chronic skin disorder that affects one in fifty people world wide and over five million people in the United States. Approximately 150,000 to 250,000 new cases of psoriasis are diagnosed each year. Ten percent of people with psoriasis develop psoriatic arthritis. The most common form of the disease is called plaque psoriasis or psoriasis vulgaris. Other forms are pustular, guttate, inverse, and erythrodermic psoriasis.
The cause of psoriasis is unknown. The skin lesions of psoriasis vulgaris are in part a result of an excessive rapid growth and turnover of keratinocytes. Current types of treatment achieve some temporary relief (e.g., steroids, anthralin, calcipotriene, coal tar with or without light therapy, psoralen with UVA treatment, methotrexate, retinoids with or without UV light, and cyclosporin A). However, harmful side effects of these treatments exist which vary in degree of health hazard, and some must be carefully monitored by a physician. Furthermore most of these treatments result in a recurrence of the psoriasic symptoms.
Ballaun et al. (J. Invest. Dermatol. (1995) 104:7-10) have shown that the three major splice forms of VEGF are produced by human epidermal keratinocytes and can be detected in the supernatant of cell cultures. In addition, VEGF upregulation has been observed in bullous pemphigoid, dermatitis herpetiformis, and erythema multiforme (Brown et al. (1995) J. Invest Dermatol. 104:744-749). Furthermore, psoriasic lesions express elevated levels of transforming growth factor alpha (TGFxcex1), a known inducer of VEGF in cell culture (Detmar et al. (J. Invest. Dermatol. (1995) 105:44-50), and VEGF levels of normal epidermal keratinocytes can be induced in response to elevated levels of TGFxcex1.
Thus, VEGF appears to play a principle role in many pathological states and processes related to angiogenesis and neovascularization. Regulation of VEGF expression in tissues affected by the various conditions described above could therefore be key in treatment or preventative therapies associated with such disorders.
New chemotherapeutic agents termed xe2x80x9cantisense oligonucleotidesxe2x80x9d have been developed which are capable of modulating cellular and foreign gene expression (see, Zamecnik et al. (1978) Proc. Natl. Acad. Sci. (USA) 75:280-284). Without being limited to any theory or mechanism, it is generally believed that the activity of antisense oligonucleotides depends on the binding of the oligonucleotide to the target nucleic acid (e.g. to at least a portion of a genomic region, gene or mRNA transcript thereof), thus disrupting the function of the target, either by hybridization arrest or by destruction of target RNA by RNase H (the ability to activate RNase H when hybridized to RNA).
VEGF-specific antisense oligonucleotides have been developed (Uchida et al. (1995) Antisense Res. and Dev. 5(1):87 (Abstract OP-10); Nomura et al., (1995) Antisense Res. and Dev. 5(1):91 (Abstract OP-18)), although none have been demonstrated to reverse neovascularization or angiogenesis. Thus, a need still remains for the development of oligonucleotides that are capable of reducing VEGF expression, and ultimately, of inhibiting the onset of diseases and disorders associated with the expression of VEGF.
It is known that cells affected by hypoxia induce VEGF, and that aberrant expression of VEGF has been observed in skin diseases characterized by neoangiogenesis and epidermal alterations. The present invention provides novel synthetic oligonucleotides specific for nucleotides 58 to 90 of the VEGF gene which can reduce the hypoxia- or TGFxcex1-induced expression of VEGF mRNA and protein. This information has been exploited to develop the present invention which includes VEGF-specific oligonucleotides, pharmaceutical formulations, and methods of reducing the expression of VEGF mRNA and protein and of treating various diseases characterized by the over-expression of VEGF.
In one aspect, the invention provides a synthetic oligonucleotide complementary to a nucleic acid specific for human vascular endothelial growth factor. This oligonucleotide has a nucleic acid sequence set forth in the Sequence Listing as SEQ ID NOS:1-16.
As used herein, the term xe2x80x9csynthetic oligonucleotidexe2x80x9d refers to chemically synthesized polymers of nucleotides covalently attached via at least one 5xe2x80x2 to 3xe2x80x2 internucleotide linkage. In some embodiments, these oligonucleotides contain at least one deoxyribonucleotide, ribonucleotide, or both deoxyribonucleotides and ribonucleotides. In another embodiment, the synthetic oligonucleotides used in the methods of the invention are from about 15 to about 30 nucleotides in length. In preferred embodiments, these oligonucleotides contain from about 16 to 29 nucleotides.
For purposes of the invention, the term xe2x80x9coligonucleotide sequence that is complementary to a genomic region or an RNA molecule transcribed therefromxe2x80x9d is intended to mean an oligonucleotide that binds to the nucleic acid sequence under physiological conditions, e.g., by Watson-Crick base pairing (interaction between oligonucleotide and single-stranded nucleic acid) or by Hoogsteen base pairing (interaction between oligonucleotide and double-stranded nucleic acid) or by any other means including in the case of a oligonucleotide binding to RNA, causing pseudoknot formation. Binding by Watson-Crick or Hoogsteen base pairing under physiological conditions is measured as a practical matter by observing interference with the function of the nucleic acid sequence.
In some embodiments, the synthetic oligonucleotide of the invention are modified in a number of ways without compromising their ability to hybridize to nucleotide sequences contained within the mRNA for VEGF. The term xe2x80x9cmodified oligonucleotidexe2x80x9d as used herein describes an oligonucleotide in which at least two of its nucleotides are covalently linked via a synthetic linkage, i.e., a linkage other than a phosphodiester linkage between the 5xe2x80x2 end of one nucleotide and the 3xe2x80x2 end of another nucleotide in which the 5xe2x80x2 nucleotide phosphate has been replaced with any number of chemical groups. In some preferred embodiments, at least one internucleotide linkage of the oligonucleotide is an alkylphosphonate, phosphorothioate, phosphorodithioate, phosphate ester, alkylphosphonothioate, phosphoramidate, carbamate, carbonate, phosphate triester, acetamidate, and/or carboxymethyl ester.
The term xe2x80x9cmodified oligonucleotidexe2x80x9d also encompasses oligonucleotides having at least one nucleotide with a modified base and/or sugar, such as a 2xe2x80x2-O-substituted ribonucleotide. For purposes of the invention, the term xe2x80x9c2xe2x80x2-O-substitutedxe2x80x9d means substitution of the 2xe2x80x2 position of the pentose moiety with an xe2x80x94Oxe2x80x94 lower alkyl group containing 1-6 saturated or unsaturated carbon atoms, or with an xe2x80x94O-aryl or allyl group having 2-6 carbon atoms, wherein such alkyl, aryl or allyl group may be unsubstituted or may be substituted, e.g., with halo, hydroxy, trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy, carboxyl, carbalkoxyl, or amino groups; or with a hydroxy, an amino or a halo group, but not with a 2xe2x80x2-H group. In some embodiments the oligonucleotides of the invention include four or five ribonucleotides 2xe2x80x2-O-alkylated at their 5xe2x80x2 terminus (i.e., 5xe2x80x2 2-O-alkylated ribonucleotides), and/or four or five ribonucleotides 2xe2x80x2-O-alkylated at their 3xe2x80x2 terminus (i.e., 3xe2x80x2 2-O-alkylated ribonucleotides). In preferred embodiments, the nucleotides of the synthetic oligonucleotides are linked by a or at least one phosphorothioate internucleotide linkage. The phosphorothioate linkages may be mixed Rp and Sp enantiomers, or they may be stereoregular or substantially stereoregular in either Rp or Sp form (see Iyer et al. (1995) Tetrahedron Asymmetry 6:1051-1054).
In another aspect, the invention provides a method of inhibiting VEGF expression. In this method, nucleic acid specific for VEGF is contacted with an oligonucleotide of the invention. As used herein, the term xe2x80x9cnucleic acidxe2x80x9d encompasses a genomic region or an RNA molecule transcribed therefrom. In some embodiments, the nucleic acid is mRNA.
Without being limited to any theory or mechanism, it is generally believed that the activity of oligonucleotides used in accordance with this invention depends on the hybridization of the oligonucleotide to the target nucleic acid (e.g. to at least a portion of a genomic region, gene or mRNA transcript thereof), thus disrupting the function of the target. Such hybridization under physiological conditions is measured as a practical matter by observing interference with the function of the nucleic acid sequence. Thus, a preferred oligonucleotide used in accordance with the invention is capable of forming a stable duplex (or triplex in the Hoogsteen pairing mechanism) with the target nucleic acid; activate RNase H thereby causing effective destruction of the target RNA molecule, and in addition is capable of resisting nucleolytic degradation (e.g. endonuclease and exonuclease activity) in vivo. A number of the modifications to oligonucleotides described above and others which are known in the art specifically and successfully address each of these preferred characteristics.
Also provided by the present invention is a pharmaceutical composition comprising at least one synthetic oligonucleotide of claim 1 in a physiologically acceptable carrier.
Another aspect of the invention includes pharmaceutical compositions capable of inhibiting neovascularization and thus are useful in the methods of the invention. These compositions include a synthetic oligonucleotide of the present invention which specifically inhibits the expression of vascular endothelial growth factor and a physiologically and/or pharmaceutically acceptable carrier.
The term xe2x80x9cpharmaceutically acceptablexe2x80x9d means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s). The term xe2x80x9cphysiologically acceptablexe2x80x9d refers to a non-toxic material that is compatible with a biological system such as a cell, cell culture, tissue, or organism.
Another aspect of the invention is assessment of the role of VEGF in neovascularization and angiogenesis associated with normal development and in various disease states.
Yet another aspect is a method of treating psoriasis by administering to a human afflicted with the disorder a therapeutically-affective amount of an oligonucleotide of the invention.
The subject oligonucleotides and methods of the invention also provide a means of examining the function of the VEGF gene in a cell, or in a control mammal and in a mammal afflicted with a neovascularization-related or psoriasis-related disorder. The cell or mammal is administered the oligonucleotide, and the expression of VEGF mRNA or protein and/or proteins which are known to interact with CDK4 is examined. Presently, gene function is often examined by the arduous task of making a xe2x80x9cknock outxe2x80x9d animal such as a mouse. This task is difficult, time-consuming and cannot be accomplished for genes essential to animal development since the xe2x80x9cknock outxe2x80x9d would produce a lethal phenotype. The present invention overcomes the shortcomings of this model.